Abstract
We determined the complete genome sequence of Clostridium difficile strain 630, a virulent and multidrug-resistant strain. Our analysis indicates that a large proportion (11%) of the genome consists of mobile genetic elements, mainly in the form of conjugative transposons. These mobile elements are putatively responsible for the acquisition by C. difficile of an extensive array of genes involved in antimicrobial resistance, virulence, host interaction and the production of surface structures. The metabolic capabilities encoded in the genome show multiple adaptations for survival and growth within the gut environment. The extreme genome variability was confirmed by whole-genome microarray analysis; it may reflect the organism's niche in the gut and should provide information on the evolution of virulence in this organism.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
McDonald, L.C. et al. An epidemic, toxin gene-variant strain of Clostridium difficile . N. Engl. J. Med. 353, 2433–2441 (2005).
Loo, V.G. et al. A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality. N. Engl. J. Med. 353, 2442–2449 (2005).
Voth, D.E. & Ballard, J.D. Clostridium difficile toxins: mechanism of action and role in disease. Clin. Microbiol. Rev. 18, 247–263 (2005).
Wust, J., Sullivan, N.M., Hardegger, U. & Wilkins, T.D. Investigation of an outbreak of antibiotic-associated colitis by various typing methods. J. Clin. Microbiol. 16, 1096–1101 (1982).
Nolling, J. et al. Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum . J. Bacteriol. 183, 4823–4838 (2001).
Shimizu, T. et al. Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater. Proc. Natl. Acad. Sci. USA 99, 996–1001 (2002).
Bruggemann, H. et al. The genome sequence of Clostridium tetani, the causative agent of tetanus disease. Proc. Natl. Acad. Sci. USA 100, 1316–1321 (2003).
Farrow, K.A., Lyras, D. & Rood, J.I. Genomic analysis of the erythromycin resistance element Tn5398 from Clostridium difficile . Microbiology 147, 2717–2728 (2001).
Haraldsen, J.D. & Sonenshein, A.L. Efficient sporulation in Clostridium difficile requires disruption of the sigmaK gene. Mol. Microbiol. 48, 811–821 (2003).
Braun, V. et al. A chimeric ribozyme in Clostridium difficile combines features of group I introns and insertion elements. Mol. Microbiol. 36, 1447–1459 (2000).
Burrus, V., Pavlovic, G., Decaris, B. & Guedon, G. Conjugative transposons: the tip of the iceberg. Mol. Microbiol. 46, 601–610 (2002).
Mullany, P. et al. Genetic analysis of a tetracycline resistance element from Clostridium difficile and its conjugal transfer to and from Bacillus subtilis . J. Gen. Microbiol. 136, 1343–1349 (1990).
Wang, H. et al. Characterization of the ends and target sites of the novel conjugative transposon Tn5397 from Clostridium difficile: excision and circularization is mediated by the large resolvase, TndX. J. Bacteriol. 182, 3775–3783 (2000).
Franke, A.E. & Clewell, D.B. Evidence for a chromosome-borne resistance transposon (Tn916) in Streptococcus faecalis that is capable of “conjugal” transfer in the absence of a conjugative plasmid. J. Bacteriol. 145, 494–502 (1981).
Roberts, A.P., Johanesen, P.A., Lyras, D., Mullany, P. & Rood, J.I. Comparison of Tn5397 from Clostridium difficile, Tn916 from Enterococcus faecalis and the CW459tet(M) element from Clostridium perfringens shows that they have similar conjugation regions but different insertion and excision modules. Microbiology 147, 1243–1251 (2001).
Garnier, F., Taourit, S., Glaser, P., Courvalin, P. & Galimand, M. Characterization of transposon Tn1549, conferring VanB-type resistance in Enterococcus spp. Microbiology 146, 1481–1489 (2000).
Jansen, R., Embden, J.D., Gaastra, W. & Schouls, L.M. Identification of genes that are associated with DNA repeats in prokaryotes. Mol. Microbiol. 43, 1565–1575 (2002).
Mojica, F.J., Diez-Villasenor, C., Garcia-Martinez, J. & Soria, E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J. Mol. Evol. 60, 174–182 (2005).
Makarova, K.S., Grishin, N.V., Shabalina, S.A., Wolf, Y.I. & Koonin, E.V. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol. Direct 1, 7 (2006).
Calabi, E. et al. Molecular characterization of the surface layer proteins from Clostridium difficile . Mol. Microbiol. 40, 1187–1199 (2001).
Wright, A. et al. Proteomic analysis of cell surface proteins from Clostridium difficile . Proteomics 5, 2443–2452 (2005).
Waligora, A.J. et al. Characterization of a cell surface protein of Clostridium difficile with adhesive properties. Infect. Immun. 69, 2144–2153 (2001).
Savariau-Lacomme, M.P., Lebarbier, C., Karjalainen, T., Collignon, A. & Janoir, C. Transcription and analysis of polymorphism in a cluster of genes encoding surface-associated proteins of Clostridium difficile . J. Bacteriol. 185, 4461–4470 (2003).
Hennequin, C., Janoir, C., Barc, M.C., Collignon, A. & Karjalainen, T. Identification and characterization of a fibronectin-binding protein from Clostridium difficile . Microbiology 149, 2779–2787 (2003).
Poilane, I., Karjalainen, T., Barc, M.C., Bourlioux, P. & Collignon, A. Protease activity of Clostridium difficile strains. Can. J. Microbiol. 44, 157–161 (1998).
Dramsi, S., Trieu-Cuot, P. & Bierne, H. Sorting sortases: a nomenclature proposal for the various sortases of Gram-positive bacteria. Res. Microbiol. 156, 289–297 (2005).
Mazmanian, S.K., Ton-That, H., Su, K. & Schneewind, O. An iron-regulated sortase anchors a class of surface protein during Staphylococcus aureus pathogenesis. Proc. Natl. Acad. Sci. USA 99, 2293–2298 (2002).
Borriello, S.P., Welch, A.R., Barclay, F.E. & Davies, H.A. Mucosal association by Clostridium difficile in the hamster gastrointestinal tract. J. Med. Microbiol. 25, 191–196 (1988).
Davies, H.A. & Borriello, S.P. Detection of capsule in strains of Clostridium difficile of varying virulence and toxigenicity. Microb. Pathog. 9, 141–146 (1990).
Depardieu, F., Bonora, M.G., Reynolds, P.E. & Courvalin, P. The vanG glycopeptide resistance operon from Enterococcus faecalis revisited. Mol. Microbiol. 50, 931–948 (2003).
Arthur, M., Depardieu, F., Molinas, C., Reynolds, P. & Courvalin, P. The vanZ gene of Tn1546 from Enterococcus faecium BM4147 confers resistance to teicoplanin. Gene 154, 87–92 (1995).
Champion, O.L. et al. Comparative phylogenomics of the food-borne pathogen Campylobacter jejuni reveals genetic markers predictive of infection source. Proc. Natl. Acad. Sci. USA 102, 16043–16048 (2005).
Selmer, T. & Andrei, P.I. p-Hydroxyphenylacetate decarboxylase from Clostridium difficile. A novel glycyl radical enzyme catalysing the formation of p-cresol. Eur. J. Biochem. 268, 1363–1372 (2001).
Elsden, S.R., Hilton, M.G. & Waller, J.M. The end products of the metabolism of aromatic amino acids by clostridia. Arch. Microbiol. 107, 283–288 (1976).
Begley, M., Gahan, C.G. & Hill, C. The interaction between bacteria and bile. FEMS Microbiol. Rev. 29, 625–651 (2005).
Sleator, R.D., Wemekamp-Kamphuis, H.H., Gahan, C.G., Abee, T. & Hill, C.A. PrfA-regulated bile exclusion system (BilE) is a novel virulence factor in Listeria monocytogenes . Mol. Microbiol. 55, 1183–1195 (2005).
Paredes, C.J., Alsaker, K.V. & Papoutsakis, E.T. A comparative genomic view of clostridial sporulation and physiology. Nat. Rev. Microbiol. 3, 969–978 (2005).
Moir, A., Corfe, B.M. & Behravan, J. Spore germination. Cell. Mol. Life Sci. 59, 403–409 (2002).
Broussolle, V. et al. Molecular and physiological characterisation of spore germination in Clostridium botulinum and C. sporogenes . Anaerobe 8, 89–100 (2002).
Carter, G.P., Purdy, D., Williams, P. & Minton, N.P. Quorum sensing in Clostridium difficile: analysis of a luxS-type signalling system. J. Med. Microbiol. 54, 119–127 (2005).
Whitehead, N.A., Barnard, A.M., Slater, H., Simpson, N.J. & Salmond, G.P. Quorum-sensing in Gram-negative bacteria. FEMS Microbiol. Rev. 25, 365–404 (2001).
Lee, A.S. & Song, K.P. LuxS/autoinducer-2 quorum sensing molecule regulates transcriptional virulence gene expression in Clostridium difficile . Biochem. Biophys. Res. Commun. 335, 659–666 (2005).
Ohtani, K., Hayashi, H. & Shimizu, T. The luxS gene is involved in cell-cell signalling for toxin production in Clostridium perfringens . Mol. Microbiol. 44, 171–179 (2002).
Lyon, G.J. & Novick, R.P. Peptide signaling in Staphylococcus aureus and other Gram-positive bacteria. Peptides 25, 1389–1403 (2004).
van Schaik, W. & Abee, T. The role of sigmaB in the stress response of Gram-positive bacteria–targets for food preservation and safety. Curr. Opin. Biotechnol. 16, 218–224 (2005).
de Vries, Y.P. et al. Deletion of sigB in Bacillus cereus affects spore properties. FEMS Microbiol. Lett. 252, 169–173 (2005).
Wilson, K.H. Efficiency of various bile salt preparations for stimulation of Clostridium difficile spore germination. J. Clin. Microbiol. 18, 1017–1019 (1983).
Bell, K.S. et al. Genome sequence of the enterobacterial phytopathogen Erwinia carotovora subsp. atroseptica and characterization of virulence factors. Proc. Natl. Acad. Sci. USA 101, 11105–11110 (2004).
Rutherford, K. et al. Artemis: sequence visualization and annotation. Bioinformatics 16, 944–945 (2000).
Carver, T.J. et al. ACT: the Artemis comparison tool. Bioinformatics 21, 3422–3423 (2005).
Acknowledgements
We would like to acknowledge the support of the Wellcome Trust Sanger Institute core sequencing and informatics groups. We thank D. Gerding and G. Songer for provision of C. difficile strains, F. Barbut and J. Emerson for help with the antibiotic susceptibility tests, and the BUGs microarray facility at St. George's Hospital for provision of the C. difficile 630 microarray. This work was supported by the Wellcome Trust.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Table 1
Features of the C. difficile CRISPRs. (PDF 60 kb)
Supplementary Table 2
Antibiotic susceptibility of C. difficile strain 630. (PDF 50 kb)
Rights and permissions
About this article
Cite this article
Sebaihia, M., Wren, B., Mullany, P. et al. The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome. Nat Genet 38, 779–786 (2006). https://doi.org/10.1038/ng1830
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/ng1830
